The development of multicellular organisms requires the orchestration of many precisely coordinated events involving cell-type specific growth, proliferation, differentiation, migration, and cell death. Not surprisingly, intercellular communication plays critical roles in these processes. Although the molecular mechanisms involved in this communication are in general poorly understood, this research field is characterized by increasingly rapid progress initiated by the realization that viral oncogenes are, in many cases, transformed versions of cellular genes (proto-oncogenes) that participate in the intercellular communication directing development. Furthermore, it has been established that many non-viral forms of cancer also result from transformation of genes involved in signal transduction (e.g. growth factors, growth factor receptors, and transcription factors).
A large number of mammalian growth factor receptors have been cloned and many are recognized proto-oncogenes (Yarden and Ullrich, 1988). Most of these cloned receptors are members of a superfamily of integral membrane proteins with intrinsic, growth factor-inducible, tyrosine kinase activity. An extensive research literature now documents the critical roles these receptors play in cell proliferation, differentiation, and malignant transformation. However, multiple lines of evidence suggest that members of the G-protein coupled receptor (GPR) superfamily may also participate in mammalian development and oncogenesis. For example, both the yeast S. cerevisiae and the slime mold D. discoideum express GPRs that regulate cell differentiation (Devreotes, 1989; Sprague, 1991). In addition, mammalian mitogenesis and cell proliferation are affected by several peptides and neurotransmitters which are known to interact with GPRs (Hanley, 1989; Zachary et al., 1987).
Perhaps the most direct evidence linking GPRs with ontogeny and cancer has been provided by the ectopic expression of GPRs in tissue culture cells. Thus, the mas oncogene encodes a putative GPR (pmas) and leads to malignant transformation when transfected into NIH3T3 mouse fibroblasts cells (Young et al., 1986). In addition, several serotonin and muscarinic acetylcholine receptors (all GPRs) also produce this malignant transformation if ectopically expressed in NIH3T3 cells and stimulated by their respective ligands (Gutkind et al., 1991; Julius et al., 1989; Julius et al., 1990). While these data illustrate that GPRs can greatly influence cell proliferation and morphology, the GPRs that were studied are unlikely to be involved in these processes in vivo because they reside in fully differentiated, postmitotic cells such as neurons where serotonergic receptors, muscarinic receptors, and most likely pmas regulate the changing electrical properties of neuronal membranes involved in neurotransmission. However, these data support the possibility that other GPRs are expressed in vivo in immature cells where they regulate proliferation and differentiation. Furthermore, these data suggest that some forms of cancer may result from mutations or viral infections that lead to improper functioning, activation, or expression of such GPRS. Thus, identification and characterization of such receptors should significantly advance both the study of normal development as well as the search for diagnostic and therapeutic tools in oncology.
The subject invention concerns the cloning and sequencing of cDNAs and the proteins encoded by those cDNAs. The cDNAs encode novel polypeptides that are members of the G-protein coupled receptor (GPR) superfamily. The proteins encoded by the DNAs of the subject invention are involved in the regulation of cell proliferation and/or differentiation in vivo. The subject protein receptors are endogenously expressed in various tissues and cell lines.
Specifically, the subject invention concerns the cloning and sequencing of a rat cDNA (H218) that encodes a novel GPR designated pH218. Further included in the subject invention are mammalian homologs, including the human homolog of the H218 cDNA. The H218 cDNA was used to determine that H218 mRNA is expressed in all developing organs tested and in seven out of seven cell lines tested. In addition, in the brain, H218 mRNA is much more highly expressed during a period of extensive proliferation and differentiation (embryogenesis) than a period of very limited cell proliferation and differentiation (adulthood), suggesting that pH218 does not function as a neurotransmitter receptor. Rather, pH218 functions as a growth factor ligand receptor.
The subject invention further concerns antibodies from animals immunized with peptides derived from pH218 GPR. Purified antibody made against one of the peptides recognizes a protein having an apparent molecular weight of 50-55 kDA as determined by Western blot analysis.
The subject invention also concerns cDNA of the rat-edg gene. Rat-edg cDNA encodes a GPR, prat-edg. The prat-edg can be activated by some of the same ligand(s) that activate pH218. By identifying compounds that specifically activate or inhibit this class of receptors one can develop unique, pharmaceutical therapies that effectively treat some forms of cancer.
A further aspect of the subject invention concerns polynucleotide molecules that are antisense to mRNA of H218 and rat-edg. The antisense polynucleotide molecules can be used to reduce or inhibit the expression of the subject protein by binding to the complementary mRNA transcripts.
The subject invention also concerns methods of use for the polynucleotide sequences, the encoded proteins, peptide fragments thereof, polynucleotide molecules that are antisense to the H218 and rat-edg sequences, and antibodies that bind to the proteins and peptides. Such use includes diagnostic and therapeutic applications of the subject invention.